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  • C12N15/00—Mutation or genetic engineering; DNA or RNA concerning genetic engineering, vectors, e.g. plasmids, or their isolation, preparation or purification; Use of hosts therefor
  • C12N15/09—Recombinant DNA-technology
  • C12N15/11—DNA or RNA fragments; Modified forms thereof; Non-coding nucleic acids having a biological activity
  • C12N15/117—Nucleic acids having immunomodulatory properties, e.g. containing CpG-motifs
• • A—HUMAN NECESSITIES
  • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
  • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
  • A61K31/00—Medicinal preparations containing organic active ingredients
  • A61K31/70—Carbohydrates; Sugars; Derivatives thereof
  • A61K31/7088—Compounds having three or more nucleosides or nucleotides
  • A61K31/713—Double-stranded nucleic acids or oligonucleotides
• • A—HUMAN NECESSITIES
  • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
  • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
  • A61K48/00—Medicinal preparations containing genetic material which is inserted into cells of the living body to treat genetic diseases; Gene therapy
• • A—HUMAN NECESSITIES
  • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
  • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
  • A61P35/00—Antineoplastic agents
• • A—HUMAN NECESSITIES
  • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
  • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
  • A61P35/00—Antineoplastic agents
  • A61P35/04—Antineoplastic agents specific for metastasis
• • A—HUMAN NECESSITIES
  • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
  • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
  • A61P37/00—Drugs for immunological or allergic disorders
  • A61P37/02—Immunomodulators
• • A—HUMAN NECESSITIES
  • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
  • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
  • A61P37/00—Drugs for immunological or allergic disorders
  • A61P37/02—Immunomodulators
  • A61P37/04—Immunostimulants
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  • C12N2310/00—Structure or type of the nucleic acid
  • C12N2310/10—Type of nucleic acid
  • C12N2310/17—Immunomodulatory nucleic acids
• • C—CHEMISTRY; METALLURGY
  • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
  • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
  • C12N2310/00—Structure or type of the nucleic acid
  • C12N2310/50—Physical structure
  • C12N2310/51—Physical structure in polymeric form, e.g. multimers, concatemers
• • C—CHEMISTRY; METALLURGY
  • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
  • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
  • C12N2310/00—Structure or type of the nucleic acid
  • C12N2310/50—Physical structure
  • C12N2310/53—Physical structure partially self-complementary or closed
• • C—CHEMISTRY; METALLURGY
  • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
  • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
  • C12N2320/00—Applications; Uses
  • C12N2320/30—Special therapeutic applications
  • C12N2320/31—Combination therapy

Definitions

• the invention relates to a polymeric, non-coding nucleic acid molecule for the modulation of the activity of the human and animal immune system as well as a method for the manufacture thereof and a vaccine, comprising the polymeric, non-coding nucleic acid molecule, wherein polymeric, non-coding nucleic acid molecules may be understood as non- coding nucleic acid molecules, comprising at least four covalently bound molecules (tetramer) of said non-coding nucleic acid molecules.
• the adaptive immune response starts with a delay (3 - 5 days) after selection of the specific lymphocytes for the respective pathogen, their clonal expansion and differentiation to effector cells, but then provides a long lasting protection for the respective pathogen by forming an immunological memory
• the cells of the innate immune system recognize pathogens via conserved pathogen associated molecular patterns (PAMP) by germ cell encoded receptors and react immediately.
• PAMP pathogen associated molecular patterns
• Different reactions belong to different kinds of cell types like the secretion of cytokines (e.g. IL-I, IL-6, TNF- ⁇ ) and chemokines (e.g.
• IL- 8/CXCL8, MlP-l ⁇ / ⁇ , MCP-I the activation of effectors mechanisms (phagocytosis, respiratory discharge, liberation of bactericide or cytotoxic substances or lytic granules), the expression of co-stimulatory molecules (CD80, CD86) as well as the enhanced expression of MHC-molecules.
• effectors mechanisms phagocytosis, respiratory discharge, liberation of bactericide or cytotoxic substances or lytic granules
• co-stimulatory molecules CD80, CD86
• the cells of the adaptive immune system receive the necessary signals for their activation.
• CpG-oligonucleotides have been used as a new class of immune modulating molecules.
• Such non-methylated CG-motives can be found in bacterial DNA and represent a "danger signal" for the immune system.
• PAMP pathogen associated molecular pattern
• CpG-ODN induce via the cytokines interleukine-12, interferon- ⁇ and tumor necrosis factor- ⁇ a T H l-based immune response.
• Immune stimulatory nucleic acids comprising said CpG-ODN, have a length of several bases and comprise no open reading frame for the expression of proteins.
• the ISS represent linear nucleic acid molecules, which ends are open (free hydroxyl- and phosphate groups) or protected by synthetic groups.
• the monomeric nucleic acids used as starting material were heated before ligation, receiving uniform molecules of nucleic acids, each consisting of a dumbbell- shaped dimer (compare figure 1 of the WO 01/07055).
• the resulting nucleic acid is a dumbbell according to figure 1 of WO 01/07055.
• Immune modulation means in the context of the present invention that the mediator and effector cells of the immune system, thus mainly the presently known thymocytes with helper function and the cytotoxic thymocytes, B-cells and so called NK (natural killer)-cells, macrophages and monocytes as well as dendritic cells and their precursors, as well as cell populations with so far not clearly identified functions which have a function within the immune system, are stimulated by the use of nucleic acid molecules for proliferation, migration, differentiation or their activity.
• Immune modulation means, that besides a general improvement of the immune response in the above defined sense also the type or character of the immune reaction will be influenced, whether by affecting a beginning or maturing immune reaction or by changing an established reaction with regard to their character.
• the molecule with improved immunemodulatory properties claimed by the present invention is compared with the dimeric substances from the WO 01/07055 a polymeric, non- coding nucleic acid molecule.
• a polymeric nucleic acid molecule should be understood as so- called high molecular concatemer.
• the invented polymeric molecule can be manufactured by a method, comprising the following steps:
• Concatemers comprise covalently bound monomer units, which are in their entirety circularly closed, having within the distance of the constitutive monomers double stranded parts and immunemodulatory CG-motives preferably in the single stranded parts. It was completely surprising, that these polymers comprising tetramers, hexamers or high molecular assemblies of covalently closed immunemodulartory DNA have a surprisingly improved effect in comparison to dimeric molecules from the state of the art.
• the claimed polymeric molecules are shown in figures 1 and 2 with regard to the molecular properties and in Figures 3 und 4 with regard to the functional properties, which result from the application of the method for the manufacture for a person skilled in the art.
• the use of nucleic acid molecules with palindromic 5'- respectively 3 '-ends as educts in the described method results in polymers with different sizes, from which only the claimed tetramers or high molecular assemblies fulfill the highly potent function. Since the characterization by structural features is not feasible due to the extended and diverse molecule size, the characterization of the polymers via their method of manufacture is very precise.
• the new method provides a different product than the one described in the state of the art. This can be demonstrated by clear differences in the properties of the dimers and polymers according to the invention as shown in figure 3.
• the high molecular polymers according to the invention are surprisingly better suited for immune modulation then the non-polymeric structures known from the state of the art.
• the molecules according to the invention can also be manufactured by providing 5'- phosphorylated deoxyribonucleotide acids in water, if they are purified with an equivalent method to a polyacrylamide gel electrophoresis, especially by the combined purification with HPLC followed by FPLC. It is known by a person skilled in the art, that by the combination of several high performance methods like HPLC or FPLC an analogue grade of purification to a PAGE-purification can be obtained.
• a mulitmeric molecule comprising circular stably and covalently with each other bound monomers with at least 24 nucleotides.
• the simultaneously formed high molecular polymers comprise always a even number of monomeric components.
• the formed chain of molecules does not comprise free 5'- or 3' ends.
• the monomers forming via intermolecular esterification the molecule according to the invention are characterized by:
• - modified nucleotides can also be part of a single stranded area, which are covalently linked to fatty acids, sugars or amino acids.
• a molecule according to the invention comprises at least four monomers and is formed with regard to its conformation during the above-mentioned synthesis.
• the monomers are forming via intermolecular bonds to catena of two, four, six or more by formation of covalent bond. This results in the formation of so called di-, tetra- or hexamers, which are all designated as polymers with exception of the dimers.
• a molecule according to the invention can be also defined as concatemer.
• the molecule according to the invention is a concatermeric molecule, wherein at least four loops of individual monomers are linked with each other, preferably linear so that preferably two especially preferred several double - stranded parts are each separated from each other by single stranded loop elements.
• a molecule according to the invention is able to modulate the activity of the human or animal immune system better compared to molecules of the state of the art.
• the molecules from the state of the art are the known immune modulatory nucleic acid sequences, which are effective as low molecular dumbbell- shaped structures.
• the most known, immune modifying short oligodeoxyribonucleotide acids comprise an unmethylated cytosine-guanosine-motive.
• a physiological effect of such nucleic acids is also understood as immune modulation respectively modulation of the activity of the immune system within the sense of the invention.
• the EP 1 196 178 discloses additionally several molecules, consisting of a stem with at least one loop, as they are disclosed for example in the figures 1 and 2 of EP 1 196 178.
• Such molecules are dimeric structures.
• the present invention does not comprise such dimers.
• polymer is used with several different meanings in science.
• a polymer may be for instance a longer nucleic acid as well as a structure comprising several of the same or similar molecules formed to a larger assembly.
• An polymer within the sense of the invention designates catena of molecules, comprising at least four monomers. If the preferred monomers are used, the molecular weight of the resulting tetramer corresponds to about 170 kDa (comp. fig. 2).
• Polymers within the sense of the invention would be for instance several stem-loop-structures as depicted in fig.
• a polymer 1, assembling with several of the same or similar stem-loop- structures to a higher structure (a polymer).
• polymer are all molecules according to the invention designated which are larger than 23 kDa.
• the described conditions for the reaction cause during the ligation a transient attachment of the monomers, which can be esterified by ligase.
• a resulting polymer will be formed during the synthesis with respect to its confirmation only under the special reaction conditions. It is not possible to manufacture the high molecular polymers from dimers that have already been formed.
• the monomer structures forming the polymer are covalently linked to each other.
• a formed polymer is stabile with respect to heat or denaturing agents, which means vice versa that the dimers can not be obtained with simple physical means out of a high molecular molecule according to the invention.
• P, W, S are nucleic acids, linked to each other in the listed reading order via phosphodiester links "-",
• sequence of the nucleic acid P, W or S comprises at least one motive of the deoxyribonucleotide sequence CG.
• W is at least 4 nucleotides long and
• sequences of the nucleic acid parts S and P are reverse complementary to each other.
• nucleic acid P at the right side in the formula is covalently linked to the left nucleic acid W
• the nucleic acid group W may comprise in this connection molecules with the sequences of B, U, K, Y
• the nucleic acid group P may comprise molecules with the sequences of J, E, R, G
• the nucleic acid group S may comprise sequences of M, A, T, I.
• sequence parts J-U-A respectively R-Y-I are present, which do not have to be sequence identical with regard to each other what is shown by index "i" respectively "n-i+1".
• the claimed polymers with sequence identical or sequence different monomer components conform to the formula:
• A, B, E, G, I, J, K, M, R, T, U, Y are deoxyribonucleotide molecules and
• the sequence of component i of a nucleic acid molecule may be different, but does not have to, compared to the (i+1) of the same molecule and
• At least one nucleic acid comprises a motive with the deoxyribonucleotide sequence
• G is covalently linked via a phosphodiester bound to B.
• a polymer according to the invention is characterized in that the deoxyribonucleic acid used in the method comprises the following sequence:
• the monomer comprises the base sequence N 1 N 2 CGN 3 N 4 , wherein N 1 N 2 is an element of the group of GT, GG, GA, AT or AA, N 3 N 4 is an element of the group CT or TT, as well as C deoxycytosine, G deoxyguanosine, A deoxyadenosine and T deoxythymidine.
• the base sequence N 1 N 2 CGN 3 N 4 is positioned within the single stranded part of the closed chain of deoxyribonucleotides. Especially these preferred molecules show very strong effects during modulation of the immune systems.
• a molecule according to the invention may have one or more substitutes bound via covalent bonds.
• substitutes may be e.g. peptides, proteins, saccharides, lipids, antigenic structures, DNA and/or RNA.
• the invention relates besides the above mentioned structural and functional features of the product also to a method for the manufacture of the molecule comprising the following steps: - providing a 5'- phosphorylated DNA molecule in water purified by polyacrylamide gel electrophoresis,
• a polymer according to the invention comprises preferably 2+2 monomers (comp. fig 1), preferred partly single stranded, covalently closed chains of deoxyribonucleotide components, wherein the monomers have a stem and a loop, wherein the stem has at least 2 deoxyribonucleotides and the loop at least 4 deoxyribonucleotides and the loop has 1 to 6 CG- motives and the variable n is an element from the set of all natural numbers.
• the invention relates further to a composition, which comprises at least a molecule according to the invention and a chemo therapeutic. It was surprising that the unexpected strong improvement of the immune response by a molecule according to the invention could be further clearly improved by combining the remedy according to the invention with known chemo therapeutic s and using the composition preferably for instance for the treatment of tumours. Although it was known by a person skilled in the art, that dimers according to WO 01/07055 have an immune modulatory effect and it was further known that chemo therapeutics have an effect on tumours, it was surprising that the immune modulatory dimers composed of monomers cause in combination with chemo therapeutics an over-additive effect.
• a composition according to the invention may be provided as a kit, in which a molecule according to the invention and the chemo therapeutics according to the state of the art are provided separately.
• the at least two components of the kits may be applied simultaneously or time delayed.
• composition according to the invention may for instance activate the immune system so that a subsequent application of a chemotherapeutic may have a very good effect. It is a matter of course, that it is possible to apply at first the chemotherapeutic and subsequently with a time delay a molecule according to the invention into the human or animal organism. For defined tumours the simultaneous application of a molecule according to the invention and the chemotherapeutic is preferred.
• a chemotherapeutic is selected from the group comprising antibodies, alkylating agents, platinum analoga, intercalating agents, antibiotics, mitosis suppresses, taxanes, topoisomerases suppressors, anti-metabolites and/or L-asparaginase, hydroxycarbamide, mitotanes and/or amanitines.
• alkylating agents are selected from the group comprising
• the alkylating agents have a very good effect on tumours, inhibiting their growth.
• platinum analoga are selected from a group comprising:
• intercalating agents are selected from the group comprising:
• antibiotics are selected from the group comprising:
• the mitoses suppressers are to selected form the group comprising: alkaloids of vinca rosea, especially
• taxanes are selected from the group comprising:
• the toposimerase suppressors are selected from the group comprising:
• topoisomerase-II-inhibitors especially,
• anit- metabolites are selected from the group comprising:
• the invention relates further to a kit, comprising the molecule according to the invention and the chemo therapeutic, if applicable together with information about the combination of the content of the kit.
• the invention relates also - as already described - to a pharmaceutical comprising the molecule according to the invention or the composition if applicable with a pharmaceutical compatible carrier.
• the invention relates further to the use of the molecule, the composition or the pharmaceutical for the manufacture of a remedy for the modulation of a human or animal immune system or for the modulation of the activity of the mentioned immune system.
• Modulation of the human or animal immune system shall be understood as each influence on the immune system, having the effect that the immune system inhibits tumours or cancer.
• the modulation of the activity of the immune system can synonymously be understood to this or be described for a person skilled in the art as the known activities of the immune system that are directed against tumours and being surprisingly increased in their effect by remedies according to the invention.
• the modulation is especially a stimulation or an increase of effects of the immune system respectively the immune system itself meaning a tumour-suppressive or remitting prophylactic effect.
• a remedy according to the invention can be used in a preferred embodiment to stimulate the T-cell mediated immune response but also to change a T-cell independent immune response.
• This process may comprise in a preferred embodiment of the invention a proliferation of B-cells or B-cell activation.
• the modulation of the activity of the immune system results in an improvement with the effect that the secretion of cytokines of different relevant cell populations is changed respectively reverted. It may be especially preferred that the molecule according to the invention respectively the composition according to the invention are used as adjuvant in therapeutic or prophylactic vaccination.
• the remedy according to the invention may be used very efficiently for the treatment of cell growth disorders, wherein in a preferred embodiment the cell growth disorder is a tumour disease.
• tumour disease is a disease selected from the group comprising tumours of the ear-nose-throat region, comprising tumors of the inner nose, nasal sinus, nasopharynx, lips, oral cavity, oropharynx, larynx, hypopharynx, ear, salivary glands, and paragangliomas, tumors of the lungs comprising non-parvicellular bronchial carcinomas, parvicel- hilar bronchial carcinomas, tumors of the mediastinum, tumors of the gastrointestinal tract, comprising tumors of the esophagus, stomach, pancreas, liver, gallbladder and biliary tract, small intestine, colon and rectal carcinomas and anal carcinomas, urogenital tumors comprising tumors of the kidneys, ureter, bladder, prostate gland, urethra, penis and testicles, gynecological tumors comprising tumors of the cervix, vagina, vulva, uterine
• ODN 5 -phosphorylated oligodeoxyribonucleotide
• the degree of polymeristaion can only be influenced to a certain degree by the concentration of the employed nucleic acid.
• the method of manufacture was modified as follows:
• Nucleic acids with the sequence S'-pCTAGGGGTTACCACCTACAAAAAAA AACGAAATTCGGGGCGAAGGGAGGTGGTAACCC-S 1 (SEQ ID No. 7) with a concentration of 1 mg/ml was precipitated with 0,3M sodium-acetate (pH 5,25), 1OmM MgCI 2 and a threefold volume of ethanol abs.. After centrifugation (4 0 C, 13000rpm) the ODN was dried at 50 0 C for lOmin. The pellet was directly used for ligation (0,5 U/ ⁇ g ODN) and incubated for 60 min at 37 0 C. Separation of the ligation product on a 3% agarose gel, compare fig. 2, lane 4.
• the manufactured molecules were separated on a3% agarose gel.
• the left lane 1 shows the molecular weights of double stranded DNA indicating the mass of each band, corresponding to the different migration distances.
• Lanes 2 to 4 were loaded with products of the different polymerisation reactions. A single band can be observed corresponding to the dimmer (Iane2) respectively the tetramer (Iane3) respectively to a ladder comprising all forms of polymers (lane 4).
• the potency of the polymeric molecules according to the invention is increased by a factor of 10 (upper curve with closed symbols) in comparison to the low molecular weight molecules (lower dashed curve with open symbols).
• High molecular polymers according to the invention have a clearly better effect with equivalent amounts used as comparable amounts of dimeric or monomeric molecules.
• the higher potency for TLR9 stimulation can be attributed to a locally higher concentration achieved by the multimeric molecules which can especially in vivo not be achieved by higher doses, e.g. for reasons of the applicable amount.
• Simultaneously the high molecular concatemers have an increased efficiency which is completely surprising and can not be explained according to the current knowledge of sciences.
• peripheral mononuclear blood cells PBMC
• the isolated cells PBMC were seeded in multi- well-plates.
• the first mixture contained not stimulated cells as negative control, the second mixture was stimulated with dimers as comparison to the state of the art, the third with tetrameric polymers; the same mass of dimmers respectively polymers was used in the same volume.
• ELISA determined the secretion of the cytokines interferon- ⁇ , interferon- ⁇ and interleukin-6 from the cell culture supernatant two days later, compare fig. 4.
• the stimulation of PBMCs with the polymeric molecules according to the invention results in a doubling of the interferon secretion in comparison to the stimulation by dimeric molecules.
• the deoxyribonucleic acid sequences used as educts are not heated prior to ligation and have a purification grade comparable to polyacrylamide electrophoresis.
• the educts can be purified by HPLC followed by FPLC.
• HPLC HPLC
• FPLC FPLC
• the DNA-educts are lyophilised at 5O 0 C until a dry residue is obtained.
• a resuspension in a buffer is made and T4- DNA ligase is added followed by an incubation at 37 0 C for 40 minutes. It was surprising, that the obtained concatemers cause an improved immune modulation in mice.
• chemotherapeutic antibodies alkylating agents, platinum analoga, intercalating agents, antibiotics, mitosis suppresses, taxanes, topoisomerases suppressors, anti-metabolites and/or L-asparaginase, hydroxycarbamide, mitotanes and/or amanitines may be used.

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